The present study have evaluated the anti-inflammatory properties of BzSA in RAW 264.7 cells as compared to a well known anti-inflammatory agent ASA. BzSA (Benzoylsalicylic acid) is first time purified from G.rottlerifirmis seed coats (Kamatham et al., 2016) where as ASA (Acetylsalicylic acid) a syntheric analogue of SA (Salicylic acid; Fig. 1). BzSA develops plants immunity and induced disease resistance in tobacco and Arabidopsis against TMV virus plants (Kamatham et al., 2016). Further more various chemically synthesized analogues of BzSA have shown a powerfull defence resistance against TMV virus even at low concentrations (Samuel et al, 2017). Inorder to deterimine the stability of BzSA We treated SA deficient Arabidopsis NahG plants with BzSA and check the defense response and surprisingly we were still able to see the defense response even in the absence of SA which indicates that BzSA is a stable molecule and does not undergo deacylation in the plants after treatment (Samuel et al 2016).
The cytotoxicity MTT assay results of RAW264.7 cells were shown that the IC50 value of BzSA slight higher than aspirin (Fig. 2). Aspirin indued a decrease in cell viability in a time and deose dependent manner (Bellosillo et al., 1998). Interestingly, BzSA have shown more COX-2 enzyme inhibition activity over ASA (Table 1). NSAIDs including aspirin, indomethacin and ibuprofen are non-selective inhibitors of COX-1 and COX-2, whereas, celecoxib and rofecoxib selectively inhibit COX-2 enzyme activity (Rao and Knaus, 2008). NSAIDs are potent anti-inflammatory and anti-cancer agents that acts through the inactivation of the COX enzymes, mostly COX-2 and thus directed the prostaglandins (PGs) synthesis at the site of inflammation (Rao and Knaus, 2008; Willoughby et al., 2000). BzSA have no effect on 5-LOX enzyme inhibition (Table 1). In view of the importance of dual COX-2/5-LOX, identification of specific inhibitors of COX-2/5-LOX proteins have therapeutic advantage (Fiorucci et al., 2001; Ranjbar et al., 2016). Interestingly, BzSA inhibits COX-2 and 5-LOX expression higher than aspirin (Fig. 3). COX-2 expression is highly restricted and selectively induced by pro-inflammatory cytokines at the site of inflammation and thus involved in the production of pro-inflammatory PGs (Crofford, 1997; Rao and Knaus, 2008; Seibert and Masferrer, 1994).
NF-κB induce 5- LOX enzyme expression during the inflammation process (Pahl, 1999). 5-LOX enzyme catalyzes AA metabolism in the leucocytes (Steinhilber, 1999). Leukotrienes (LTs) are the metabolic products of AA metabolism, which possess a potent pro-inflammatory activity and thus might be involved in cardiovascular diseases (de Gaetano et al., 2003). Decreased expression of 5-LOX gene enhanced cell death in breast cancer cells and therefore plays an essential role in reducing the tumor proliferation (Kumar et al., 2016). The 5-LOX enzyme inhibitor zileuton reduces inflammatory reaction in the ischemic brain damage through the activation of P13K/Akt signaling pathway (Tu et al., 2016). A report suggest that 7-subsituted coumarin derivatives are potential 5-LOX inhibitors (Srivastava et al., 2016).
Interestingly, the inhibition effect of BzSA on COX-1 enzymatic activity very low as compared to ASA (Table 1). However, BzSA and ASA have no effect at COX-1 expression even increasing concentrations (Fig. 3). One of the major disadvantage of ASA is inhibition of COX-1 and associated anti-thrombic and ulcerogenic side effects (Bianchi Porro and Pace, 1988; Undas et al., 2007). COX-1 expressed constitutively in almost all cell types in the human body and thus produced anti-inflammatory PGs which are important to maintain the homeostatic functions such as integrity of the gastric mucosa, platelet function, and renal blood flow (Allison et al., 1992). These results encouraging that the dentification of selective inhibitors of COX-2 with out effecting COX-1 are useful therapeutics.
In order to determine that BzSA is a potent inhibitor of NF-KB pathway we are pre-treated Raw 264.7 cells with BzSA and analysed the IKK-α/IKK-β an essential up-stream signaling of NF-NF-KB. Surprisingly, BzSA down-regulated IKK-α/β expression higher than ASA (Fig. 4). NF-κB pathway is under the control of IKK-kinase complex. IKKα/β phosphorylats IκB-α an inhibitor of NF-κB (Adli et al., 2010; Aggarwal, 2006; Karin and Delhase, 2000) and reduced phosphorylation of IκB and NF-κB pathway. ASA binds to IKK-β and inhibit ATP binding and phosphorylation of IκB (Aggarwal, 2006; Yin et al., 1998). In our results we also observed the similar action of BzSA on IKKα/β (Fig. 4). Phosphorylation of IκB-α by IKKα/β brings IκB-α-NF-κB inactive complex to active form and leading to the activation and localization of NF-κB in to nucleous (Aggarwal, 2006; Huang and Hung, 2013; Yamamoto and Gaynor, 2001; Yin et al., 1998). A dose wise IκBα inbibition and reduction in p-NF-κB p65 was observed in BzSA higher than ASA treatment and supported by previous observations (Fig. 4) (E.Britta-Mareen Traenckner et al., 1995; Vancurova and Vancura, 2012). NF-κB-DNA binding subunits are released and translocated into the nucleus thereby activates a wide range of inflammation responsive genes (Hayden and Ghosh, 2008; Rao and Knaus, 2008; Vallabhapurapu and Karin, 2009). Benzo[b]thiophen-2-yl-3-bromo-5-hydroxy-5H-furan-2-one (BTH) inhibited NF-κB and STAT3 phosphorylation (Andres et al., 2013). Isoorientin is a natural flavone reduced NF-κB in RAW 264.7 cells (Sumalatha et al., 2015). NF-κB recognized as a key player involved in many steps of cancer initiation and progression (Hoesel and Schmid, 2013; Karin, 2009).
In order to validate the effect of BzSA on NF-κB pathway we tested a series of NF-κB targeted inflammatory mediaters. BzSA suppressed COX-2 (Fig. 3 ). COX- 2 expression depends on NF-κB activation (Lim et al., 2001). Previosely it was reported that pro and anti-inflammatory nature of COX-2 (Poligone and Baldwin, 2001). The expression of inflammatory mediators such as TNF-α, COX-2, 5-LOX, iNOS and cytokines such as IL-1β and IL-6 require translocation of NF-κB from cytosol to nucleus (Rao and Knaus, 2008). TNF-α is a pleiotropic cytokine, which is a therapeutic target for inflammatory diseases therefore TNF-α inhibitors are therapeutic advantages in inflammatory disorders and malignant diseases (Esposito and Cuzzocrea, 2009; Parameswaran and Patial, 2010). Previously, it was reported that ASA inhibits TNF-α gene expression in RAW 264.7 cells through the suppression of active NF-κB binding to the TNF-α promotor (Shackelford et al., 1997). No significant inhibition of TNF-α by BzSA and ASA noticed (Fig. 5). Literature suggested that induced expression of TNF-α activated by NF-κB in different cell lines (Ke et al., 2007; Parameswaran and Patial, 2010; Wajant et al., 2003). Similarly, the expression of iNOS depends on the nuclear localization of activated NF-κB. BzSA suppressed iNOS higher than ASA (Fig. 5). Previously, it was suggested that inducible nitric oxide synthase (iNOS) is activated by several immunological stimuli and the resultant accumulated nitric oxide cytotoxic to the cells. Furthermore, iNOS mutants exhibited reduced immune response to carrageenin and showed resistance to LPS induced mortality (Vig et al., 2004; Wei et al., 1995). Increased iNOS associated with malignant diseases, particularly malignant transformation, angiogenesis and metastasis (Lechner et al., 2005).
Interestingly, in our study we observed more suppression of IL-1β in BzSA treated cells as compared to ASA (Fig. 5). Interleukins are the cytokines important in the regulation of immune responses, inflammatory reactions and hematopoiesis. Over activation of inmmune system causes several inflammatory disorders. Anti-inflammatory agents including ASA, anti-cytokine therapies, small molecules and many drugs under clenical trails. IL-1β and IL-6 are the the pro-inflammatory cytokines responds to inflammation. IL-1β induces IL-6 in peripheral blood monocytes (Tosato and Jones, 1990). Pro-inflammatory cytokines such as TNF-α, IL-1β and vascular growth factor (VEGF) plays a central role ininflammation (Dinarello, 2010). In our results we also noticed remarkable reduction in IL-6 by BzSA in a dose dependent manner over ASA (Fig. 5). Previously reports demonstrated that IL-1β and TNF-α induce IL-6 and most of the biological activities associated with IL-1 and IL-6 (Tosato and Jones, 1990).